Department of Biochemistry, Hacettepe University, Sihhiye, Ankara, Turkey.
Protein J. 2010 May;29(4):250-6. doi: 10.1007/s10930-010-9246-4.
Glutathione reductase [GR, E.C.1.8.1.7] catalyses NADPH dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH). Thus, it is the crucial enzyme to maintain high [GSH]/[GSSG] ratio and physiological redox status in cells. Kidney and liver tissues were considered as a rich source of GR. In this study, rat kidney GR was purified and some of its properties were investigated. The enzyme was purified 2,356 fold with a yield of 16% by using heat-denaturation and Sephadex G25 gel filtration, 2',5'-ADP Agarose 4B, PBE94 column chromatographies. The purified enzyme had a specific activity (Vm) of 250 U/mg protein and the ratio of absorbances at wavelengths of A (273)/A (463,) A (280)/A (460), A (365)/A (460), and A (379)/A (463), were 7.1, 6.8, 1.2 and 1.0, respectively. Each mol of GR subunit bound 0.97 mol of FAD. NADH was used as a coenzyme by rat kidney GR but with a lower efficiency (32.7%) than NADPH. Its subunit molecular weight was estimated as 53 kDa. An optimum pH of 6.5 and optimum temperature of 65 degrees C were found for rat kidney GR. Its activation energy (Ea) and temperature coefficient (Q(10)) were calculated as 7.02 kcal/mol and 1.42, respectively. The Km((NADPH)) and kcat/Km ((NADPH)) values were found to be 15.3 +/- 1.4 microM and 1.68 x 10(7) M(-1) s(-1) for the concentration range of 10-200 microM NADPH and when GSSG is the variable substrate, the Km((GSSG)) and the kcat/Km((GSSG)) values of 53.1 +/- 3.4 microM and 4.85 x 10(6) M(-1) s(-1) were calculated for the concentration range of 20-1,200 microM GSSG.
谷胱甘肽还原酶[GR，EC1.8.1.7]催化 NADPH 依赖的将谷胱甘肽二硫化物（GSSG）还原为还原型谷胱甘肽（GSH）。因此，它是维持细胞中高[GSH]/[GSSG]比和生理氧化还原状态的关键酶。肾脏和肝脏组织被认为是 GR 的丰富来源。在这项研究中，我们纯化了大鼠肾脏 GR 并研究了其一些性质。该酶通过使用热变性和 Sephadex G25 凝胶过滤、2'，5'-ADP 琼脂糖 4B、PBE94 柱层析，以 2356 倍的纯度和 16%的产率进行纯化。纯化后的酶具有 250 U/mg 蛋白的比活性（Vm），波长 A（273）/A（463）、A（280）/A（460）、A（365）/A（460）和 A（379）/A（463）的吸光度比值分别为 7.1、6.8、1.2 和 1.0。每个 GR 亚基结合 0.97 mol 的 FAD。NADH 被大鼠肾脏 GR 用作辅酶，但效率较低（32.7%），而不是 NADPH。其亚基分子量估计为 53 kDa。发现大鼠肾脏 GR 的最适 pH 为 6.5，最适温度为 65°C。其活化能（Ea）和温度系数（Q(10)）分别计算为 7.02 kcal/mol 和 1.42。当 GSSG 为可变底物时，发现 NADPH 浓度范围为 10-200 microM 时，Km((NADPH))和 kcat/Km((NADPH))值分别为 15.3 +/- 1.4 microM 和 1.68 x 10(7) M(-1) s(-1)，而 GSSG 的 Km((GSSG))和 kcat/Km((GSSG))值分别为 53.1 +/- 3.4 microM 和 4.85 x 10(6) M(-1) s(-1)，GSSG 浓度范围为 20-1200 microM。
